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EGF Stimulates Rab35 Activation and Gastric Cancer Cell Migration by Regulating DENND1A-Grb2 Complex Formation.

ABSTRACT: Aims: The aim of this study was to reveal the specific molecular mechanisms by which DENND1A accepts EGF signaling and activates Rab35 in gastric cancer. Methods: The expression of proteins related to DENND1A was examined by western blot analysis. Activation of Rab35 was assessed by GST-pulldown. The interaction of DENND1A and Grb2 was assessed by GST-pulldown and co-immunoprecipitation assays. The relationship between DENND1A and cell migration and invasion was detected using wound healing and transwell by gene overexpression and RNA interference. Results: EGF stimulation significantly promoted cell migration, whereas transfection with siRab35 partially inhibited EGF-promoted cell migration. DENND1A is also involved in these processes and active Rab35. Moreover, DENND1A binds to the N-terminal and C-terminal SH3 domains of Grb2 through PRD. Of special interest is the observation that EGFR can recruit Grb2-DENND1A complex under EGF stimulation. Further results reveal that the higher the expression of DENND1A, the shorter progression-free survival of gastric cancer patients. Conclusion: In summary, we confirmed that EGF-Grb2-DENND1A-Rab35 signaling pathway with the interaction of DENND1A and Grb2 as a regulatory center could regulate gastric cancer cell migration and invasion. Ultimately, the expression level of DENND1A predicts the survival status of gastric cancer patients and may become one of the important targets for the treatment of gastric cancer.

SUBMITTER: Ye B

PROVIDER: S-EPMC6261971 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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EGF Stimulates Rab35 Activation and Gastric Cancer Cell Migration by Regulating DENND1A-Grb2 Complex Formation.

Ye Bixing B Duan Biao B Deng Wenjie W Wang Yueyuan Y Chen Yan Y Cui Jie J Sun Shixiu S Zhang Yujie Y Du Jun J Gu Luo L Lin Lin L Tang Yurong Y

Frontiers in pharmacology 20181122

<b>Aims:</b> The aim of this study was to reveal the specific molecular mechanisms by which DENND1A accepts EGF signaling and activates Rab35 in gastric cancer. <b>Methods:</b> The expression of proteins related to DENND1A was examined by western blot analysis. Activation of Rab35 was assessed by GST-pulldown. The interaction of DENND1A and Grb2 was assessed by GST-pulldown and co-immunoprecipitation assays. The relationship between DENND1A and cell migration and invasion was detected using woun ...[more]

PMID: 30524285

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Project description:MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3'-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.

Dataset Information

EGF Stimulates Rab35 Activation and Gastric Cancer Cell Migration by Regulating DENND1A-Grb2 Complex Formation.

Publications

EGF Stimulates Rab35 Activation and Gastric Cancer Cell Migration by Regulating DENND1A-Grb2 Complex Formation.

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